Compositions and methods for treatment of pain

ABSTRACT

The present invention provides compositions and methods for treatment or prevention of pain resulting from infection, infection related pain, and non-infectious pain as well as treatment or prevention of infections or adverse health consequences associated with infections.

This application claims priority to provisional patent application Ser.No. 61/450,937, filed Mar. 9, 2011, which is herein incorporated byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbers R01DK066112 and T32 DK062716-05 by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention provides compositions and methods for treatment orprevention of pain resulting from infection, infection related pain, andnon-infectious pain as well as treatment or prevention of infections oradverse health consequences associated with infections.

BACKGROUND OF THE INVENTION

Urinary tract infection (UTI) is the second most common infectiousdisease that sends both men and women to seek treatment. In the UnitedStates, UTI accounts for >7 million office visits and >1 millionemergency room visits, necessitating 100,000 hospitalizations annually(Schappert S M. Vital Health Stat 1997; 13:1-38; herein incorporated byreference in its entirety). Most community-acquired UTIs are due toinfection by uropathogenic Escherichia coli (UPEC) that elicit aninflammatory response in the bladder during acute bacterial cystitis.Patients with UTI frequently have symptoms that include dysuria, voidingfrequency or urgency, and pelvic pain. In contrast, ˜5% of patients withUTI do not exhibit any of these symptoms and receive a diagnosis ofasymptomatic bacteriuria (ASB) (Nicolle et al., Clin Infect Dis 2005;40:643-654; herein incorporated by reference in its entirety). Althoughmost patients with UTI experience pelvic pain, the mechanism underlyingUTI-induced pelvic pain remains unknown.

UTIs are generally treated with antibiotics as a first line oftreatment. Drugs most commonly recommended for simple UTIs includeamoxicillin (Amoxil, Trimox), ciprofloxacin (Cipro), nitrofurantoin(Furadantin, Macrodantin), trimethoprim (Proloprim) and the antibioticcombination of trimethoprim and sulfamethoxazole (Bactrim, Septra). Forsevere UTIs, hospitalization and treatment with intravenous antibioticsmay be necessary. However, many antibiotic resistant bacteria arepresent in the environment, especially in hospital and other health caresettings. Thus, additional treatments for UTIs are needed.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for treatment orprevention of pain resulting from infection, infection related pain, andnon-infectious pain as well as treatment or prevention of infections oradverse health consequences associated with infections.

In some embodiments, the present invention provides a method fortreatment or prevention of pain comprising administering a probioticcomposition to a subject having pain (e.g., pelvic pain). In someembodiments, the present invention provides a method for treatment orprevention of pain arising from infection (e.g., active or resolved UTIinfection caused by E. coli), treatment of active infection (e.g.,decrease in number of infectious organisms), treatment of infectionrelated pain, treatment of non-infectious pain (e.g., chronic or acutenon-infectious pain), or treatment of symptoms of an infection (e.g.,non-pain symptom), comprising administering a probiotic composition.

In some embodiments, probiotic compositions comprise probiotic bacteria.In some embodiments, probiotic compositions comprise compositionsderived from probiotic bacteria. In some embodiments, compositions arederived directly from probiotic bacteria (e.g., isolated and/or purifiedfrom bacteria). In some embodiments, compositions are derived indirectlyfrom probiotic bacteria (e.g., synthetically or recombinantly producedcompositions). In some embodiments, probiotic compositions comprise LPS.In some embodiments, probiotic compositions comprise peptides,carbohydrates, lipids, nucleic acids, and/or other organic molecules. Insome embodiments, compositions comprise asymptomatic E. coli strainsisolated from the human urinary tract include, but are not limited to,NUA2, NUA1, 2-21, 2-37, 2-35, 2-32, 2-13, 2-29, 2-42, 2-44, 2-8, 2-43,NUA3, 83972, 2-33 or 2-12. In some embodiments, probiotic bacteriacomprise E. coli strain 83972, 2-12 or a bacteria or strain thatproduces a functionally equivalent reduction in pain.

In some embodiments, pain is treated or prevented through aTLR4-dependent pathway. In some embodiments, pain is treated orprevented through an inflammation-independent pathway. In someembodiments, a probiotic composition treats or prevents infection and/orinflammation (e.g., in addition to treating pain). In some embodiments,a probiotic composition treats or prevents pain without treating orpreventing infection and/or inflammation. In some embodiments, aprobiotic composition is administered as a tablet, capsule, pill,injection, cream, ointment, lotion, slave, balm, suppository, solution,elixir, syrup, suspension, cream, lozenge, paste or spray. In someembodiments, a probiotic composition is administered systemically. Insome embodiments, a probiotic composition is administered locally to theregion of infection and/or pain (e.g., vaginally). In some embodiments,a probiotic composition is administered such that pain in the affectedregion of infection and/or pain is treated via organ crosstalk or viacorresponding dermatomes. In some embodiments, the present inventionprovides a composition that reduces pain resulting directly orindirectly from infections in a subject.

In some embodiments, the present invention provides a compositioncomprising a probiotic composition formulated for treating pain (e.g.,pelvic pain related or unrelated to infection by pathogenic bacteria).In some embodiments, a probiotic composition comprises a probioticbacteria. In some embodiments, a probiotic compositions comprises acomposition derived from a probiotic bacteria. In some embodiments,compositions comprise asymptomatic E. coli strains isolated from thehuman urinary tract include, but are not limited to, NUA2, NUA1, 2-21,2-37, 2-35, 2-32, 2-13, 2-29, 2-42, 2-44, 2-8, 2-43, NUA3, 83972, 2-33or 2-12. In some embodiments, probiotic bacteria comprise E. coli strain83972, 2-12 or a bacteria or strain that produces a functionallyequivalent reduction in pain. Additional embodiments are describedherein.

DEFINITIONS

As used herein, the term “probiotic” refers to microorganisms that havebeneficial effects to a subject they are administered to. A probioticmay be administered live. “Probiotic compositions” may refer to aprobiotic organism, a portion thereof, or a composition derived from aprobiotic organism.

As used herein, the term “asymptomatic E. coli strain isolated from thehuman urinary tract” refers to a bacteria (e.g., probiotic E. colibacteria) present in the human urinary tract that does not cause pain orother symptoms of an infection. Examples of asymptomatic E. coli strainsisolated from the human urinary tract include, but are not limited to,NUA2, NUA1, 2-21, 2-37, 2-35, 2-32, 2-13, 2-29, 2-42, 2-44, 2-8, 2-43,NUA3, 83972, 2-33 or 2-12.

As used herein, the term “pain related to infection” refers to any pain(e.g., pelvic pain) that is related to infection (e.g., infection by apathogenic bacteria such as pathogenic E. coli). In some embodiments,pain is caused by an active bacterial infection (e.g., urinary tractinfection). In other embodiments, pain related to infection is pain inan individual that has previously had a bacterial infection that hasbeen treated (e.g., the subject does not have an active infection).

As used herein, the term “non-infectious pain” refers to pain that isnot related to an active infection or is associated with a priorinfection. In some embodiments, “non-infectious pain” is “chronicnon-infectious pain” or “acute non-infectious pain.” As used herein, theterm “chronic non-infectious pain” refers to pain that persists forextended periods of time (e.g., days, weeks, months or years). In someembodiments, “chronic non-infectious pain” refers to chronic pain thathas not responded to treatment (e.g., pain medicine or anti-infectiveagent treatments).

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vivo, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and an emulsion, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants see Martin, Remington'sPharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975).

“Pharmaceutically acceptable salt” as used herein, relates to anypharmaceutically acceptable salt (acid or base) of a compound of thepresent invention, which, upon administration to a recipient, is capableof providing a compound of this invention or an active metabolite orresidue thereof. As is known to those of skill in the art, “salts” ofthe compounds of the present invention may be derived from inorganic ororganic acids and bases. Examples of acids include hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic,malonic, naphthalene-2-sulfonic and benzenesulfonic acid. Other acids,such as oxalic, while not in themselves pharmaceutically acceptable, maybe employed in the preparation of salts useful as intermediates inobtaining the compounds of the invention and their pharmaceuticallyacceptable acid.

As used herein, the term “nutraceutical,” refers to a food substance orpart of a food, which includes a fusion protein. Nutraceuticals canprovide medical or health benefits, including the prevention, treatment,or cure of a disorder.

The term “sample” as used herein is used in its broadest sense. As usedherein, the term “sample” is used in its broadest sense. In one sense itcan refer to a tissue sample. In another sense, it is meant to include aspecimen or culture obtained from any source, as well as biological.Biological samples may be obtained from animals (including humans) andencompass fluids, solids, tissues, and gases. Biological samplesinclude, but are not limited to blood products, such as plasma, serumand the like. These examples are not to be construed as limiting thesample types applicable to the present invention. A sample suspected ofcontaining a human chromosome or sequences associated with a humanchromosome may comprise a cell, chromosomes isolated from a cell (e.g.,a spread of metaphase chromosomes), genomic DNA (in solution or bound toa solid support such as for Southern blot analysis), RNA (in solution orbound to a solid support such as for Northern blot analysis), cDNA (insolution or bound to a solid support) and the like. A sample suspectedof containing a protein may comprise a cell, a portion of a tissue, anextract containing one or more proteins and the like.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of compositions andmethods that may be provided by the present invention. Typically, theterms “subject” and “patient” are used interchangeably herein inreference to a human subject.

DESCRIPTION OF THE FIGURES

FIG. 1 shows graphs depicting NU14 inducing pelvic pain in female mice.Referred visceral hyperalgesia was measured as responses to mechanicalstimulation of the pelvic region by using von Frey filaments of 5calibrated forces. Data are reported as mean percentages of positiveresponse±standard errors of the mean before instillation of bacteria(baseline) and at postinfection day (PID) 1, 2, 3, 4, 5, 6, 7, 10, and14. A, Responses to pelvic stimulation of saline-infected femaleC57BL/6J mice (B6). B, Responses to pelvic stimulation of female B6 miceinfected with 83972. C, Responses to pelvic stimulation of female B6mice infected with NU14. D, Responses to pelvic stimulation of femaleKit^(W) ^(—) ^(sh)/Kit^(W) ^(—) ^(sh) mice infected with NU14. Analysisof variance indicated a significant increase in response frequency frombaseline for all filaments tested in NU14-treated mice on days 1-10,with no significant differences in baseline between saline- andNU14-treated mice. NU14-treated mice exhibited a significant increase inresponse frequency from baseline in the largest 3 filaments only at day14. E, Percentage responses for each day calculated as total responsesto all fibers relative to baseline responses.

FIG. 2 shows graphs demonstrating that UTI-associated pelvic pain isattenuated by organ cross-talk. Modulation of referred visceralhyperalgesia by colonic lidocaine was measured as responses tomechanical stimulation of the pelvic region with von Frey filaments of 5intensities. Responsiveness was characterized before infection(baseline), on postinfection day (PID) 1, and 45 min after colonicadministration of 2% lidocaine on day 1. Instilling 50 μL of lidocaineinto the colon of B6 mice; B) or mast cell-deficient Kit^(W) ^(—) ^(sh)mice; C) reduced pelvic pain responses, whereas lidocaine had nosignificant effect on mice infected with 83972.

FIG. 3 shows graphs demonstrating that NU14-induced pelvic pain is notcorrelated with bacterial colonization of the bladder. Wild-type micewere infected with either 83972 or NU14, and bacterial colonization wasmeasured 24 h or 14 days after infection. A and C, Both 83972 and NU14colonized the bladder, but NU14 colonization was significantly greaterthan 83972 colonization at 24 h (A) and 14 days (C) after infection;cfu, colony-forming units; PID, postinfection day. Mean colonizationlevels are indicated by solid lines; bladder homogenates withoutdetectable bacterial colonization appear on the x-axis. B and D, Pelvicpain was not correlated with bladder colonization at 24 h or 14 daysafter infection.

FIG. 4 shows graphs demonstrating that Type 1 pilus status does notinfluence urinary tract infection (UTI)-associated pelvic pain. Referredvisceral hyperalgesia was measured as responses to mechanicalstimulation of the pelvic region by von Frey filaments of 5 intensities.Responsiveness was characterized at baseline and 24 h after bacterialinfection. A, Responses to pelvic stimulation of female B6 mice infectedwith 83972 without the fimB-fimD gene cluster (no type 1 pili). B,Responses to pelvic stimulation of female B6 mice infected with83972:pREG153 without the fimB-fimD gene cluster (no type 1 pili). C,Responses to pelvic stimulation of female wild-type mice infected with83972:pGB4 with the fimB-fimD gene cluster (expresses type 1 pili). D,Responses to pelvic stimulation of wild-type mice infected with NU14with fimH (expresses type 1 pili). E, Responses to pelvic stimulation ofwild-type mice infected with NU14-1 without fimH (greatly reducedexpression of type 1 pili). F, Percentage responses 24 h after infectionwere calculated as total responses to all fibers relative to baselineresponses for 83972 (white bar), 83972:pREG153 (light gray bar),83972:pGB4 (dark gray bar), and NU14 with or without fimH (black bars).

FIG. 5 shows graphs demonstrating that NU14-induced pelvic pain is notcorrelated with urine neutrophil myeloperoxidase (MPO). Female B6 micewere infected with 83972, 83972:pGB4, NU14, or NU14-1, and neutrophilMPO, a measure of inflammation was quantified 6 h or 24 h afterinfection. A, NU14-1 MPO levels are significantly lower than those forboth NU14 and 83972:pGB4 6 h after infection, with no otherstatistically significant differences between any groups. C, NU14-1 MPOlevels were significantly lower than those for 83972:pGB4, and NU14 MPOlevels were significantly higher than those for 83972 24 h afterinfection, with no other statistically significant differences betweenany groups C. B and D, Mean MPO levels are indicated by solid lines.Pelvic pain was not correlated with MPO levels at 6 h or 24 h afterinfection. E and F, Histograms of CD80⁺ cells 4 h (E) and 8 h (F) afterlipopolysaccharide (LPS) stimulation. Solid gray line with shading,medium; dashed line, isotype control; dotted black line, 83972; solidblack line, NU14. G, Both NU14 and 83972 LPS significantly increasednumbers of CD80⁺ macrophages compared with medium. H, Both NU14 and83972 LPS induced significantly increased levels of interleukin (IL)-6.

FIG. 6 shows graphs demonstrating that lipopolysaccharide (LPS) inducespelvic pain and represents a therapeutic target for pain relief.Referred visceral hyperalgesia was measured as responses to mechanicalstimulation of the pelvic region with von Frey filaments of 5intensities. Responsiveness was characterized at baseline and 1, 4, 24,48, 72, and 96 h after 83972 or NU14 LPS instillation. A, Responses topelvic stimulation of female B6 mice instilled with NU14 LPS. Analysisof variance indicated a significant increase in response frequency frombaseline for all filaments tested in NU14 LPS-treated mice at 1, 4, and24 h after instillation, with no significant differences in baselinebetween 83972- and NU14-treated mice. B, Percentage responses 1, 4, 24,48, 72, and 96 h after instillation were calculated as total responsesto all fibers relative to baseline responses for 83972 or NU14LPS-instilled mice. C, LPS-induced myeloperoxidase (MPO) levels in 83972and NU14 LPS-instilled mice. D, C3H-HeJ mice instilled with NU14 LPSexhibited a significant reduction in pelvic pain compared with C3H-HeOuJmice instilled with NU14 LPS. Percentage responses 4 h afterinstillation were calculated as total responses to all fibers relativeto baseline responses. E, Left panel is a cartoon and experimentaltimeline (intervention, saline or 83972 LPS). Right panel is a graph ofthe percentage responses at 1, 2, 3, 4, 5, and 6 days after NU14instillation, calculated as total responses to all fibers relative tobaseline responses for all groups of mice.

FIG. 7 shows that SΦ874-induced chronic pelvic pain is differentiallyattenuated by 83972.

FIG. 8 shows that ΔwaaL-induced chronic pelvic pain is differentiallyattenuated by 83972.

FIG. 9 shows that 2-23-induced chronic pelvic pain is attenuated by83972.

FIG. 10 shows response to NU14 induced pain with and withoutantibiotics. A. Study overview. B. Response to saline. C. Response toCiprofloxacin. D. Response to NU14. E. Response to NU14 plusCiprofloxacin. F. Pain response in the presence of saline orCiprofloxacin. G. Colonization of NU14 in the presence of saline orCiprofloxacin.

FIG. 11 shows pain response and colonization of NU14 and the presenceand absence of 83972. Pain response in presence of lidocaine (A) or83972. C and D. NU14 colonization in the presence of 83972 or saline. Eand F. 83972 colonization.

FIG. 12 shows pain response with and without treatment with a variety ofprobiotic isolates. A and B. Pain response following treating with avariety of strains. C. Pain after first, second, or third infection.

FIG. 13 shows pain response and colonization of NU14 with 2-12.

FIG. 14 shows treatment with antibiotics and 2-12. FIG. 15A shows that2-12 provides pain relief superior to Cipro. FIGS. 15B and C show that2-12 has antimicrobial activity in urine (B) and vagina (C).

FIG. 15 shows treatment with antibiotics and 2-12. A. Pain response inthe presence of saline or 2-12. B and C. NU14 colonization in thepresence of saline or 2-12. D and e. 2-12 colonization in the presenceof saline or 2-12. F. NU14 infection timecourse.

FIG. 16 shows treatment with antibiotic and 2-12. A. Pain response. B.NU14 colonization. D. Colonization in the presence of control or NU14.FIGS. 16C, E and G show that 2-12 relieves pain of Proteus, E faecalisor Klebsiella UTI that is superior to Cipro

FIG. 17 shows that 2-12 is antimicrobial against UPEC pyelonephritis (B)and bladder Proteus/Kleb/E. faec (D). A. 2-12 colonization. C.Colonization in the presence of control or NU14.

FIG. 18 shows 2-12 colonization (Saline, Cipro, 2-12). A) CFT703 B)Proteus.

FIG. 19 shows 2-12 colonization (Saline, Cipro, 2-12). A) Klebsiella B)E. faecalis.

FIG. 20 shows that oral Streptococci induce differential pain statesindependent of colonization. A) Bladder instillation of 10⁸ CFU S.anginosus caused chronic pain that was significant from baseline(P<0.05*) by Day 2 and significant from S. intermedius by Day 10(P<0.05**; n=5). S. gordonii and constellatus induced no pain response.B) Most urines (U) and bladders (B) were sterile at Day 14 and Day 40,respectively, with no correlation with pain.

FIG. 21 shows that TLRs mediate S. milleri pain. Mice (n=5) wereinstilled with S. anginosis via catheter, and pelvic pain responses werequantified at Day 2. TLR2−/− mice and TLR4−/− mice showed responsesreduced by 64% and 47%, respectively (P=0.24 and P=0.41).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for treatment orprevention of pain resulting from infection, infection related pain, andnon-infectious pain as well as treatment or prevention of infections oradverse health consequences associated with infections.

Uropathogenic Escherichia coli (UPEC) are responsible for the majorityof uncomplicated urinary tract infections, which can present clinicallyas cystitis or pyelonephritis. Infection of the urinary tract by UPEC isassociated with a robust innate immune response characterized byurothelial production of inflammatory chemokines and cytokines. Localproduction of inflammatory chemokines results in the rapid recruitmentof neutrophils into the bladder lumen, which in turn mediate bacterialclearance (Schilling et al., Escherichia coli. Proc Natl Acad Sci USA2003; 100:4203-4208; Hang et al., J Infect Dis 2000; 182:1738-1748hereinincorporated by reference in their entireties). The activation of theinnate immune response in the urinary tract is dependent on patternrecognition of UPEC pathogen-associated molecular patterns by so-calledpattern recognition receptors. UPEC pathogen-associated molecularpatterns include lipopolysaccharide (LPS), flagella, type 1 pili, andpap pili (Zhang et al., Science 2004; 303:1522-1526; Hedlund et al., MolMicrobiol 2001; 39:542-552; herein incorporated by reference in theirentireties), which can act through family members of pattern recognitionreceptors, including the Toll-like receptors (TLRs) and nucleotidebinding and oligomerization domain-like receptors (Kawai et al., Cell2007; 129:1024; herein incorporated by reference in its entirety).

The cystitis isolate NU14 is considered archetypal for UPEC and has beenused to study many aspects of UTI pathogenesis (Johnson et al., J InfectDis 2001; 184:1556-1565; herein incorporated by reference in itsentirety). Several virulence factors have been characterized in UPEC,such as type 1 pili and LPS; the best-characterized UPEC virulencefactor is the type 1 pilus, adhesive organelle (Hultgren et al., AnnuRev Microbiol 1991; 45:383-415; herein incorporated by reference in itsentirety). Type 1 pili bind host urothelial cells by virtue of the FimHadhesin protein at the pilus tip, and FimH has lectin activity specificfor mannosylated glycoproteins (Sokurenko et al., Mol Microbiol 2001;41:675-686; herein incorporated by reference in its entirety).Instillation of the UPEC isolate NU14 induced rapid urothelial apoptosisthat was abrogated after instillation of the isogenic ΔfimH mutantNU14-1, indicating a requirement for type 1 pili in the urothelialapoptotic response to UPEC infection (Mulvey et al., Science 1998;282:494-497; Klumpp et al., Infect Immun 2001; 69:6689-6695; hereinincorporated by reference in their entireties). LPS is also a commonvirulence factor and is a component of the gram-negative cell wall thatbinds to TLR4, initiating the innate response against UPEC. In culture,TLR4 signaling is required for nuclear factor κB activation and mediatesLPS induction of urothelial interleukin (IL)-8 secretion by NU14(Backhed et al., Cell Microbiol 2002; 4:493-501; Schilling et al.,Infect Immun 2003; 71:1470-1480; herein incorporated by reference intheir entireties). Despite this understanding of specific virulencefactors in UTI pathogenesis, the mechanisms of UTI-associated pain arenot understood.

Pain originating from a visceral organ is typically referred to acorresponding “dermatome” on the skin that shares common spinal cordinnervation with the given visceral organ (Sturge, Brain 1883;5:492-510, herein incorporated by reference in its entirety). Thisphenomenon has been used to show that bladder-induced pelvic pain ismast cell dependent and can be positively or negatively modulated byvisceral organ cross-talk in a murine neurogenic cystitis model (Rudicket al., PLoS ONE 2008; 3:e2096; Rudick et al., Am J Physiol Regul IntegrComp Physiol 2007;293:R1191-R1198, herein incorporated by reference intheir entireties).

In experiments conducted during development of embodiments of thepresent invention, a murine urinary tract infection (UTI) model was usedto compare pelvic pain behavior elicited by infection with uropathogenicEscherichia coli strain NU14 and ASB strains 83972 and 2-12.NU14-infected mice exhibited pelvic pain, whereas mice infected with83972 did not exhibit pain, similar to patients infected with 83972 or2-12. NU14-induced pain was not dependent on mast cells, not correlatedwith bacterial colonization or urinary neutrophils. UTI pain was notinfluenced by expression of type 1 pili, the bacterial adhesiveappendages that induce urothelial apoptosis. However, purified NU14lipopolysaccharide (LPS) induced Toll-like receptor 4 (TLR4)-dependentpain, whereas 83972 LPS induced no pain. Indeed, 83972 LPS attenuatedthe pain of NU14 infection, suggesting therapeutic potential. These dataindicate a mechanism of infection-associated pain that is dependent onTLR4, yet independent of inflammation. These findings also indicateprobiotic therapies that minimize the symptoms of infection withoutreliance on therapies that contribute to antimicrobial resistance.

Mast cell-deficient mice infected with NU14 develop the same magnitudeof pelvic pain as infected wild-type mice, suggesting that UTI pelvicpain develops independent of mast cells. Pelvic pain in the neurogeniccystitis is dependent on mast cell-derived histamine and is transducedby histamine receptors 1 and 2 (Rudick et al, 2008, supra; incorporatedherein by reference in its entirety). In contrast, pelvic pain inducedin murine UTI originates from bacterial LPS that is transduced by TLR4.Therefore, the differential role of mast cells in cystitis models may bedue to the nature of the initiating insult, which neurogenic cystitis isinitiated in the central nervous system, whereas UTI in the peripheralnervous system. The present invention is not limited to any particularmechanism of action and an understanding of the mechanism of action isnot necessary to practice the present invention.

Mast cells play roles in bladder pathophysiology that are separable frombladder-associated pain, and experiments conducted during development ofembodiments of the present invention indicate similarly separable hostresponses induced by UPEC infection. NU14 induced pelvic pain behaviorin mice that was not observed after infection with the ASB strain 83972,thus recapitulating human behavioral responses to UPEC and ASB strains(Andersson et al., Infect Immun 1991; 59:2915-2921; Lindberg U. ActaPaediatr Scand 1975; 64:718-724; incorporated herein by reference intheir entireties). FimH induces rapid urothelial apoptosis and formationof urothelial lesions, indicating that FimH bladder pathology mediatespain. However, no relationship was identified between type 1 pilusexpression and pain behavior, for either NU14 or 83972 (FIG. 4).Inflammation is often assumed to underlie infection pain, but aconsistent relationship between pain and inflammation was not observedin experiments conducted during development of embodiments of thepresent invention. Pain did decay over time as urinary MPO decayed, butboth whole bacteria and LPS purified from NU14 and 83972 induced similarinflammatory responses in vitro and in vivo. This is consistent withclinical findings that patients with ASB, who lack pain by definition,nonetheless often have pyuria (Schaeffer et al., supra; Nicolle et al.,supra). Thus, the inflammation of cystitis is not sufficient by itselfto mediate the pain of cystitis. Although LPS mediates cystitis painthrough TLR4, the effects on pain appear independent of inflammatoryactions.

Experiments conducted during development of embodiments of the presentinvention demonstrate that LPS mediates pelvic pain behavior. NU14 LPSconfers a smooth colony morphology, whereas 83972 exhibits the roughphenotype, together suggesting that O antigen mediates the painresponse. However, ECOR reference strain 71 is an ASB strain with O78serotype, indicating that O antigen alone is insufficient to endow astrain with a pain-causing phenotype.

Experiments conducted during development of embodiments of the presentinvention demonstrate that LPS isolated from NU14 induced pelvic painthrough a TLR4-dependent mechanism, providing a novel pathway of pelvicpain induction and relief. C3H-OuJ and C3H-HeJ are co-isogenic mousestrains, making them ideal for studying LPS-induced TLR4 signaling. Overtime, the C3H-HeJ strain developed a spontaneous mutation in the TLR4gene, rendering these mice defective in TLR4 signaling (35, incorporatedherein by reference in its entirety). Although TLR4 involvement is welldocumented for bacterial defense, it has only recently been implicatedin nociception. TLR4 signaling is important in patients with ASB andanimal models (Ragnarsdottir et al., J Infect Dis 2007; 196:475-484;Hagberg et al., Infect Immun 1984; 46:839-844; Fischer et al., Eur JImmunol 2006; 36:267-27737-39, incorporated herein by reference in theirentireties). Ragnarsdottir et al. (supra) report lower neutrophil TLR4expression levels in patients with ASB that correspond with elevatedlevels of the TRIF adaptor protein and reduced levels of the TLR4inhibitor SIGIRR. Experiments conducted during development ofembodiments of the present invention collectively demonstrate a role forTLR4 in UTI-induced pelvic pain.

Although the present invention is not limited to any particularmechanism of action and an understanding of the mechanism of action isnot necessary to practice the present invention, it is contemplated thatLPS triggers UTI pain peripherally or centrally. TLR4 has beenimplicated in neuropathic pain because activation of microglial TLR4leads to nuclear factor κB-dependent cyclooxygenase 2 up-regulation thatcontributes to central sensitization in peripheral injury models (Tangaet al., Proc Natl Acad Sci USA 2005; 102:5856-5861; Rhee et al., J BiolChem 2000; 275:34035-34040; Ma et al., Brain Res 2002; 937:94-99; Broomet al., Neuroscience 2004; 124:891-900; incorporated herein by referencein their entireties). Likewise, intrathecal LPS induces enhanced dorsalhorn neuronal firing that correlates with allodynia and hyperalgesia(Reeve et al., Eur J Pain 2000; 4:247-257; incorporated herein byreference in its entirety). Alternatively, TLR4 could mediate painperipherally, either through sensory nerves or via the urotheliumitself. Nociceptors have recently been shown to express TLR4, whichcould then lead to LPS-induced firing due to TLR-induced protein kinaseC activation (Wadachi et al., J Dent Res 2006; 85:49-53; Acosta et al.,J Neurosci Res 2008; 86:1077-1086; Aksoy et al., Int J Biochem Cell Biol2004; 36:183-188; Numazaki et al., J Biol Chem 2002; 277:13375-13378;Premkumar et al., Nature 2000; 408:985-990; incorporated herein byreference in their entireties). Finally, urothelial TLR4 could mediatepain responses indirectly, by stimulating urothelial production ofreactive oxygen species that can then activate peripheral nociceptors(Holthusen et al., J Physiol 1995; 487(Pt 1):253-258; incorporatedherein by reference in its entirety). The involvement of TLR4 identifiesa therapeutic target for managing UTIs. The present invention is notlimited to any particular mechanism of action and an understanding ofthe mechanism of action is not necessary to practice the presentinvention.

Current UTI treatment practice employs empirical use of antimicrobialsto eradicate acute infection, although the clinical condition of ASBalready indicates that bladder infection is not necessarily deleteriousin itself. Moreover, experimental infection of human subjectsdemonstrated that UTI was resolved naturally, over the course of severaldays, in the absence of intervention with antimicrobials (Wullt et al.Infect Dis Clin North Am 2003; 17:279-301 incorporated herein byreference in its entirety). Together, these observations provide analternative therapeutic strategy for UTIs: treat the symptoms that drivepatients to visit their physicians: pain and discomfort. Experimentsconducted during development of embodiments of the present inventiondemonstrate that LPS purified from 83972 or 2-12 attenuated pelvic painduring NU14 infection (FIGS. 6E and 10-19). Data indicate that 83972—orLPS derived from an ASB strain—suppress UTI pain by interfering withTLR4-dependent pain induced by UPEC, thereby providing a novel treatmentstrategy using a probiotic that would minimize the symptoms of infectionwithout reliance on empirical therapies that contribute to antimicrobialresistance.

Accordingly, in some embodiments, the present invention providescompositions and methods for treating pain (e.g., pain related to UTI)(e.g., TLR4-dependent pain). In some embodiments, compositions andmethod comprise compositions (e.g., probiotics or compositions derivedtherefrom, LPS, LTA, etc.) or administration of such compositions to asubject for the treatment of pain (e.g., pelvic pain) (e.g.,TLR4-dependent) pain. In some embodiments, the present inventionprovides probiotics (e.g., E. coli strain 83972 or 2-12, relatedstrains, functionally equivalent strains, etc.) for the treatment ofinfection-related pain. In some embodiments, the present inventionprovides administration of compositions derived from, or related toprobiotics (e.g., membrane fractions, LPS, functional derivatives of LPSsuch as mimetics, LTA, proteins, peptides etc.) for the treatment orprevention of infection (e.g., pain, inflammation, antimicrobialtreatment, etc.). In some embodiments, the present invention providestreatment or prevention of pain that is inflammation-independent. Insome embodiments, the present invention provides administration of LPS,LTA, or other compositions derived (e.g., directly or indirectly (e.g.,produced recombinantly, synthetically produced) from probiotic organismsfor the treatment of infection and/or pain.

In some embodiments, a treated subject does not have an infection at thetime of treatment. In some embodiments, the subject does not have aurinary tract infection, a lower urinary tract infection, and/or arecurrent lower urinary tract infection. In some embodiments, thesubject does not have a history of recurrent lower urinary tractinfection, even if no infection is present at the time or treatment. Insome embodiments, the subject does not suffer from incomplete bladderemptying. Likewise, in some embodiments, methods herein are not employedfor the treatment or prevention of such infections or signs or symptoms.

In some embodiments, the present invention provides compositions andmethods for treatment for non-infectious pain (e.g., pain in individualswith no active infection or no history of active infection). In someembodiments, the present invention provides compositions and methods fortreatment of chronic non-infectious pain.

In some embodiments, the present invention provides bacteria or otherpathogens, microbes, infectious agents, etc. which provide treatment orprevention of pain or non-infectious pain. The present invention is notlimited to a particular bacterial strain. In some embodiments, thestrain is an asymptomatic E. coli strain isolated from the human urinarytract (e.g., NUA2, NUA1, 2-21, 2-37, 2-35, 2-32, 2-13, 2-29, 2-42, 2-44,2-8, 2-43, NUA3, 83972, 2-33 or 2-12; See e.g., FIG. 12). AsymptomaticE. coli strains isolated from the human urinary tract can be obtained,for example, from the E. coli Reference Center, Wiley Laboratory, WileyLane, The Pennsylvania State University.

In some embodiments, the present invention provides E. coli strain83972, 2-12, or other functional similar or equivalent strains of E.coli or other bacteria that provide similar treatment or prevention ofpain. In some embodiments, the present invention provides E. colistrains (e.g., 83972, 2-12, or related strains). In some embodiments,the present invention provides Enterobacteriaceae, Enterobacteriales,Gammaproteobacteria, Proteobacteria, Gram-negative bacteria,gram-positive bacteria (e.g., streptococcus species such as S. milleri),LPS-producing bacteria, LTA-producing bacteria, bacteria, microbes,and/or microorganisms capable of treating or preventing pain (e.g., painarising from infection).

In some embodiments, the present invention provides compositionscontaining the bacterial strains described herein (e.g., 83972, 2-12 orrelated strains), bacterial products (e.g., LPS, peptides, LTA, etc.)derived (e.g., directly or indirectly) or obtained from the abovebacteria and microorganisms. In some embodiments, compositions arepharmaceutical compositions comprising the active agent (e.g., thebacterial strains described herein) and one or more pharmaceuticallyacceptable carriers. In some embodiments, active agents (e.g., thebacterial strains described herein) are provided as a component of anutraceutical, food or food product or as a food additive.

Those skilled in the art will appreciate that the compositions disclosedherein can be readily formulated to include additional compounds commonin the pharmaceutical arts such as, excipients, extenders,preservatives, and bulking agents depending on the intended use of acomposition. Furthermore, ingestible formulations of these compositionsmay also comprise any material approved by the United States Departmentof Agriculture (USDA) for incorporation into food products such assubstances that are generally recognized as safe (GRAS) including, foodadditives, flavorings, colorings, vitamins, minerals, andphytonutrients. The term phytonutrients as used herein, refers toorganic compounds isolated from plants having biological effectsincluding, but not limited to, compounds from the following classes ofmolecules: isoflavonoids, oligomeric proanthcyanidins, indol-3-carbinol,sulforaphone, fibrous ligands, plant phytosterols, ferulic acid,anthocyanocides, triterpenes, omega 3/6 fatty acids, polyacetylene,quinones, terpenes, cathechins, gallates, and quercitin.

In some embodiments, the present invention provides treatment orprevention of TLR4-dependent pain. However, the present invention is notlimited to pain produced by any particular target or pathway, and anunderstanding of the mechanism of the present invention is not requiredto practice methods described herein. In some embodiments, the presentinvention treats and/or prevents pain, inflammation, and/or infection bytargeting TLR4-dependant or other pathways. In some embodiments, pain,inflammation, and/or infection are treated and/or prevented by targetingpathways not involving TLR4. In some embodiments, compositions andmethods of the present invention provide a wide range of benefits. Insome embodiments, compositions and methods induce a beneficialinflammatory response. In some embodiments, beneficial results (e.g.,antimicrobial, pain reduction, etc.) are obtained without induction ofan inflammatory response. In some embodiments, the present inventionprovides modulation of inflammatory response.

The present invention is illustrated for the treatment of pelvic pain(e.g., related or unrelated to UTI). However, the present invention isnot limited to the treatment of pelvic pain. The compositions andmethods of embodiments of the present invention find use in thetreatment of a variety of infections (e.g., reduction in count ofinfectious agent), treatment of symptoms of an infection (e.g., pain ornon-pain symptoms), infection related pain, and non-infectious pain.Examples include, but are not limited to, pain of the uro-genital and GItracts (e.g., vulvodynia, interstitial cystitis/painful bladdersyndrome, urinary tract infection, bladder infection, urethralinfection, prostate infection, and irritable bowel syndrome), back pain,migraine, otitis, sinusitis, orchitis, epididymitis, specific bacterialinfections (e.g., stomach ulcers, gastritis), ear infections (e.g.,chronic ear infections), chlamydia, mastitis, endometritis, Gonococcalinfections, etc. In some embodiments, the present invention providestreatment, prevention or symptom reduction of infections caused byStaphylococcus, Streptococcus, Clostridium, Bacillus, Erysipelothrixrhusiopathae, Listeria monocytogenes, Arcanobacterium pyogenes,Actinomyces, Nocardia, Mycobacterium, Enterobacteria, Enterobacteria,Salmonella, Escheria coli, Yersinia, Klebsiella, Shigella, Pseudomonas,Bordetella, Lawsonia intracellularis, Helicobacter, Spirochaetes,Borrelia, Chlamydia, Mycoplasma, Candida albicans, etc. In someembodiments, the present invention provides treatment, prevention,and/or symptom reduction (e.g., pain or inflammation reduction) ofinfections of the bladder, kidney, urinary tract, vagina, genitals,urethra, digestive tract, respiratory system, ears, nose, nasalpassages, sinus, oral cavity, oral-facial pain related to oral cavity,eye, etc.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

EXAMPLE 1 Compositions and Methods

Animals. Adult female mice (C57BL/6J, C3H-HeOuJ, or C3H-HeJ; 10-14 weeksold) were purchased from Jackson Laboratory. Mast cell-deficient Kit^(W)^(—) ^(sh)/Kit^(W) ^(—) ^(sh) mice (B6.Cg-Kit^(W) ^(—)^(sh/HNihrJaeBsmJ); 10-14 weeks old) were maintained in facilities (17,herein incorporated by reference in its entirety). All experiments wereperformed using protocols approved by the Northwestern University AnimalCare and Use Committee.

UPEC and ASB strains. NU14 is a clinical isolate of E. coli originallyobtained from the urine of a patient with cystitis (Table 1) and isconsidered archetypal for UPEC (13, herein incorporated by reference inits entirety). NU14-1 is the corresponding fimH mutant that does notexpress type 1 pili (21, herein incorporated by reference in itsentirety). ASB strain 83972 was isolated from a young Swedish girl whowas infected for at least 3 years without symptoms (19, 20, hereinincorporated by reference in their entireties) and is one of the mostextensively characterized ASB strains (22, 23, herein incorporated byreference in their entireties).

TABLE 1 Escherichia coli Strains MSHA Strain Characteristics responseReference NU14 B2 cystitis isolate Positive [21] NU14-1 NU14 withinsertional Negative [21] disruption of fimH 83972 Asymptomaticbacteriuria Negative [19] isolate 83972:pREG153 83972 with vectorplasmid Negative [22] pREG153 83972:pGB4 83972 with fim plasmid pGB4Positive [22] NOTE. MSHA, mannose-sensitive hemagglutination.

Infection. Female mice were anesthetized with isoflurane and instilledvia transurethral catheter with a volume of 10 μL containing 1×10⁸colony-forming units of either NU14 or 83972 bacteria. After infection,bladders were harvested, homogenized, and plated on eosin methylene blueagar for colonization (24, herein incorporated by reference in theirentireties). Urine was prepared according to manufacturer'srecommendations and assayed for neutrophil myeloperoxidase (MPO) byenzyme-linked immunosorbent assay (Hycult Biosciences). Mice were testedbefore bacterial infection (baseline) and for up to 14 days afterinfection. Referred hyperalgesia and tactile allodynia were tested, asdescribed elsewhere (17, 18, herein incorporated by reference in theirentireties), using von Frey filaments applied to the abdomen (17, 18,25, herein incorporated by reference in their entireties) and theplantar region of the hind paw (17, 18, 25, herein incorporated byreference in their entireties).

Purification of LPS. LPS was isolated from E. coli strains NU14 and83972 grown overnight on Luria-Bertani agar plates. Cultures werecollected by swabbing, suspended in phosphate-buffered saline, andcollected by centrifugation. LPS was then isolated using the LPSExtraction Kit (iNtRON Biotechnology), according to the manufacturer'srecommended protocol. Preparations were then further purified to removeany contaminants that could activate additional members of the TLRpathway (26 herein incorporated by reference in its entirety); thisprocess included ethanol precipitation, digestion with DNase I andRNase, and digestion with proteinase K, followed by a final ethanolprecipitation. The concentration of LPS in each sample was determinedwith the Purpald assay, by measuring 2-keto-3-deoxyoctonate (KDO) levelsand comparing them to a KDO standard curve (27, herein incorporated byreference in its entirety).

LPS instillation. NU14 or 83972 LPS (2.0 μg/25 μl) was instilled viatransurethral catheter into the bladder while isoflurane anesthesia wasmaintained. All mice were tested for referred hyperalgesia with von Freyfilaments, before and 1, 4, 24, 48, 72, and 96 h after LPS instillation.

Lidocaine treatment. Lidocaine drug therapy was administered as a 2%lidocaine solution in water that was instilled into the colon via aHamilton syringe catheter (18, herein incorporated by reference in itsentirety).

Macrophage responses to LPS. Splenic macrophages were isolated from8-10-week-old mice using CD11b MACS in conjunction with MACS LS magneticcolumns (Miltenyi); the obtained purity was ˜85%. Purified splenicmacrophages were cultured under standard conditions in the presence of100 ng/mL 83972 or NU14 LPS for 4 or 8 h. After incubation, supernatantswere collected for quantifying secreted IL-6 by enzyme-linkedimmunosorbent assay (R&D Systems), and LPS-stimulated cells wereimmediately analyzed by flow cytometry. Flow cytometry was performed bystaining for the following antibodies: allophycocyanin-anti-CD80 orbiotin anti-CD86 and streptavidin-conjugatedphycoerythrin-indotricarbocyanine (eBioscience), phycoerythrin-CD11b,and anti-CD16-CD32; hamster IgG and rat IgG2a,κ were used as isotypecontrols (BD PharMingen). Stained cells were analyzed on a FACSCantoflow cytometer (Becton Dickinson) with FACSDiva acquisition and FlowJoanalysis software, version 8.7.3 (Tree Star).

Statistical analyses. Results were expressed as means±standard errors ofthe mean. Colonization, inflammation, and behavioral data were analyzedwith the Student t test or a Kruskal-Wallis test, followed by the Dunnpost test, or with analysis of variance, followed by Dunnett's posttest; Prism software, version 5 (GraphPad), was used, as appropriate.

EXAMPLE 2 TLR4-Dependent Pain

NU14 induction of pain specific to the pelvic area. In experimentsconducted during development of embodiments of the present invention,female C57BL/6J (B6) mice were instilled with 1×10⁸ colony-forming unitsof E. coli into the bladder via transurethral catheter (Table 1). Toassess tactile sensitivity, mice were stimulated with von Freyfilaments. Mechanical stimulation of the pelvic area of saline-instilledmice resulted in a response frequency associated with the applied force,and this response profile did not change during the 14-day course of theexperiment (FIGS. 1A and 1E). Similar to findings in saline-instilledmice, the response profile of 83972-infected mice did not changesignificantly during the 14-day course of the experiment (FIGS. 1B and1E). In contrast, although NU14-infected mice exhibited the samebaseline response as saline- and 83972-infected mice, the response topelvic stimulation was significantly greater by day 1 after infection,peaked at day 2, and slowly declined but remained significantly elevateduntil day 10 (FIGS. 1C and 1E). There were no NU14-induced changes intactile sensitivity of the plantar region of the hind paw or detectableweight changes. These data indicate that NU14 induces no changes ingross physiology and pain specific to the pelvic region.

Mast cells and NU14-induced pelvic pain. Experiments conducted duringdevelopment of embodiments of the present invention demonstrate that amouse model of interstitial cystitis developed bladder-associated pelvicpain that is dependent on mast cells (17, herein incorporated byreference in its entirety). Mast cells have been shown to be importantin defense from bacterial infection (28). To test whether mast cellsplay a role in NU14-induced pain, mast cell-deficient (Kit^(W) ^(—)^(sh)/Kit^(W) ^(—) ^(sh)) mice were infected with NU14. Similar toNU14-infected B6 mice, NU14-infected Kit^(W) ^(—) ^(sh)/Kit^(W) ^(—)^(sh) mice exhibited responses to pelvic stimulation that weresignificantly greater by day 1 after infection, peaked at day 2, andremained significantly elevated until day 10 (FIGS. 1D and 1E). Incontrast to the wild-type NU14-infected mice, NU14-infected Kit^(W) ^(—)^(sh)/Kit^(W) ^(—) ^(sh) mice exhibited a prolonged increase in pelvicsensitivity until day 7 (FIG. 1E), indicating that mast cells may helpresolve the pelvic pain.

Organ cross-talk in UTI-associated pelvic pain. Instillation of 2%lidocaine directly into the colon attenuates bladder-induced pelvic painin mice, demonstrating organ cross-talk in pelvic pain relief (18,herein incorporated by reference in its entirety). A similar strategywas used to determine whether NU14-induced pelvic pain was modulated byorgan cross-talk. Lidocaine instilled into the colon significantlyreduced the response to mechanical stimulation with von Frey filaments,by ˜66% in wild-type infected mice (FIG. 2B) and ˜56% in mastcell-deficient mice (FIG. 2C), whereas 83972 animals exhibited no lossof pelvic sensitivity after saline instillation (FIG. 2A). These datademonstrate that neurogenic cystitis and UTI-associated pelvic pain aresimilarly modulated by organ cross-talk.

NU14-induced pelvic pain not correlated with bladder colonization. Micewere infected with either 83972 or NU14, and bacterial colonization wasmeasured 24 h and 14 days after infection. Both 83972 and NU14 colonizedthe bladder, but NU14 colonization was significantly greater than 83972colonization at 24 h and 14 days after infection (FIGS. 3A and 3C).However, pelvic pain was not correlated with bladder colonization at 24h (FIG. 3B) or 14 days (FIG. 3D) after infection.

Type 1 pilus status and UTI-associated pelvic pain. A difference betweenNU14 and 83972 strains is that NU14 bacteria express type 1 pili ontheir surface, whereas 83972 bacteria are not piliated (Table 1). Pilusexpression was varied for NU14 and 83972 bacteria (Table 1), instilledthese bacteria into the bladder of B6 mice, and then assessed painresponses. Before instillation, a hemagglutination assay was performedon each bacterial strain tested to confirm the presence or absence oftype 1 pili. We found that endowing 83972 with functional type 1 pilusexpression did not induce significant pelvic pain (FIGS. 4A-C and 4F).In contrast, both NU14 (fimH⁻) and NU14-1 (fimH⁻) induced significantpelvic pain in infected mice (FIG. 4D-F). These data demonstrate thattype 1 pilus status does not influence UTI-associated pelvic pain,indicating that pain is initiated by other bacterial factors.

NU14-induced pelvic pain not correlated with bladder inflammation. MPOwas quantified as a marker of inflammation in the murine UTI model (29).MPO was also quantified to determine whether pelvic pain correlateddirectly with inflammation. Mice were infected with either 83972 orNU14, and MPO levels in the urine were measured 6 or 24 h afterinfection. MPO was not significantly different between 83972- andNU14-infected mice at 6 or 24 h after infection (FIGS. 5A and 5C,respectively). Pelvic pain was not correlated with MPO levels at 6 h(FIG. 5B) or 24 h (FIG. 5D) after infection. Furthermore, in primarycultures of macrophages, both NU14 and 83972 LPS induced similarsignificant increases in CD80⁺ cells at 4 h (FIGS. 5E and 5G), withrecovery by 8 h (FIG. 5F), whereas IL-6 supernatant levels weresignificantly elevated only at 8 h (FIG. 5H). In contrast, CD86⁺ cellnumbers were unchanged at either 4 or 8 h after NU14 or 83972 LPSapplication, compared with medium.

Induction of pelvic pain by NU14 LPS. Studies have shown that when LPSwas injected intraperitoneally or into the footpad of rats it producedhyperalgesia (30, 31, herein incorporated by reference in theirentireties), and LPS has been shown to be important in bacteria-inducedbladder inflammation (32, 33, herein incorporated by reference in theirentireties). LPS was purified from 83972 or NU14 and instilled directlyinto the bladder of female B6 mice. The response profile of 83972LPS-instilled mice did not change during the 4-day course of theexperiment (FIG. 6B). In contrast, the response to pelvic stimulationwas significantly greater 1 h after NU14 LPS instillation, peaking at 4h after instillation and declining to baseline levels by 96 h afterinstillation (FIGS. 6A and 6B). Furthermore, levels of urinary MPO 6 hafter instillation did not differ significantly between 83972 and NU14LPS-instilled mice (FIG. 6C). These data suggest that NU14 LPS iscapable of inducing pelvic pain independent of neutrophil-inducedinflammation.

LPS acts through TLR4, and TLR4 is expressed on nociceptive neurons (34,herein incorporated by reference in its entirety). TLR4-deficientC3H-HeJ and the isogenic wild-type C3H-HeOuJ mouse strain was used todetermine if NU14 LPS induces pelvic pain through TLR4. C3H-HeJ miceexhibited significantly reduced pelvic pain compared with the C3H-HeOuJmice 4 h after instillation of NU14 LPS (FIG. 6D). These data indicatethat NU14 LPS is acting through TLR4 to initiate NU14-induced pelvicpain.

83972 LPS did not cause pelvic pain. B6 mice were instilled with NU14bacteria and then instilled with saline 24 h later; Saline instillationdid not alter the development of significant pelvic pain (FIG. 6E).Similarly, mice instilled with both NU14 bacteria and 83972 LPS and theninstilled with saline 24 h later also developed significant pelvic pain.In contrast to these groups, mice instilled with NU14 bacteria and theninstilled with 83972 LPS 24 h later exhibited a 40% reduction inNU14-induced pelvic pain 24 h after instillation of 83972 LPS (FIG. 6F).These data indicate that 83972 LPS acts as a TLR4 antagonist toattenuate UTI-induced pelvic pain.

EXAMPLE 3 83972-Induced Analgesia

This example describes the analgesic activity of 83972 E. coli.

Experiments were conducted to show the benefit of 83972 against paincaused by transient SF874 infection. 83972 was administered at 20 daysafter SF874 infection. Results are shown in FIG. 7. Vaginal 83972administration confers long-lasting relief, whereas bladderadministration yields only transient benefit. A single infection withSF874 was sufficient to induce pain for weeks—equivalent to >6 yrs in ahuman.

Additional experiments were conducted to show the benefit of 83972 onpain caused experiments by DwaaL. DwaaL induces “adaptive chronic” painupon a second and third serial infection. 83972 was administered at 14days after a 3^(rd) DwaaL infection. Results are shown in FIG. 8. 83972suppresses pain transiently (bladder) or durably (vagina), dependingupon site of administration.

Experiments were conducted to show the benefit of 83972 on pain causedby 2-23. 2-23 is a clinical strain isolated from a cystitis patient, andit induces chronic pain in mice that lasts the equivalent of severalhuman years. 83972 was administered at 60 days after SF874 infection.Results are shown in FIG. 9. 83972 brings long-lasting relief.

EXAMPLE 4 2-12-Induced Analgesia

This example describes the analgesic activity of 2-12 E. coli.

A variety of probiotic E. coli strains were compared to Ciprofloxin intheir ability to reduce pain caused by a variety of pathogenic E. colistrains. Results are shown in FIGS. 10-19. The results indicate that ASBstrain 2-12 has attributes including analgesic effects, antimicrobialactivity, and effects that are applicable to UTI by UPEC as well as UTIby other uropathogens.

EXAMPLE 5 Streptococcus Probiotics

This example describes strains of Streptococcus. Results are shown inFIGS. 20-21 and show that different strains can induce acute, chronic,or null pain (asymptomatic) responses. FIG. 20 shows that oralStreptococci induce differential pain states independent ofcolonization. A) Bladder instillation of 10⁸ CFU S. anginosus causedchronic pain that was significant from baseline (P<0.05*) by Day 2 andsignificant from S. intermedius by Day 10 (P<0.05**; n=5). S. gordoniiand constellatus induced no pain response. B) Most urines (U) andbladders (B) were sterile at Day 14 and Day 40, respectively, with nocorrelation with pain.

FIG. 21 shows that TLRs mediate S. milleri pain. Mice (n=5) wereinstilled with S. anginosis via catheter, and pelvic pain responses werequantified at Day 2. TLR2−/− mice and TLR4−/− mice showed responsesreduced by 64% and 47%, respectively (P=0.24 and P=0.41).

All publications and patents cited in the present application and listedbelow are herein incorporated by reference. Various modification andvariation of the described methods and compositions of the inventionwill be apparent to those skilled in the art without departing from thescope and spirit of the invention. Although the invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the relevant fields are intended to be within the scope ofthe following claims.

1. A method for treatment or prevention of pain comprising administeringa probiotic composition to a subject having pelvic pain.
 2. The methodof claim 1, wherein said pain is due to an active or inactive urinarytract infection.
 3. The method of claim 1, wherein said subject haspreviously been diagnosed with a urinary tract infection.
 4. The methodof claim 1, wherein said probiotic compositions comprise probioticbacteria.
 5. The method of claim 1, wherein said probiotic compositionscomprise compositions derived from probiotic bacteria.
 6. The method ofclaim 1, wherein probiotic compositions comprise LPS.
 7. The method ofclaim 4, wherein said probiotic composition an asymptomatic E. colistrain isolated from the human urinary tract.
 8. The method of claim 7,wherein said probiotic composition comprises a strain of E. coliselected from the group consisting of NUA2, NUA1, 2-21, 2-37, 2-35,2-32, 2-13, 2-29, 2-42, 2-44, 2-8, 2-43, NUA3, 83972, 2-33 and 2-12. 9.The method of claim 8, wherein said probiotic bacteria comprises E. colistrain 83972 or a bacteria or strain that produces a functionallyequivalent reduction in pain.
 10. The method of claim 8, wherein saidprobiotic bacteria comprises E. coli strain 2-12 or a bacteria or strainthat produces a functionally equivalent reduction in pain.
 11. Themethod of claim 1, wherein said probiotic composition treats or preventsinfection and/or inflammation in addition to pain.
 12. The method ofclaim 1, wherein said probiotic composition treats or prevents painwithout treating or preventing infection and/or inflammation.
 13. Themethod of claim 1, wherein said probiotic composition is administered asa tablet, capsule, pill, injection, cream, ointment, lotion, slave,balm, suppository, solution, elixir, syrup, suspension, cream, lozenge,paste or spray.
 14. The method of claim 1, wherein said probioticcomposition is administered systemically.
 15. The method of claim 1,wherein said probiotic composition is administered locally to the regionof infection and/or pain.
 16. The method of claim 13, wherein saidprobiotic composition is administered vaginally.
 17. The method of claim1, wherein said probiotic composition is administered such that pain inthe affected region of infection and/or pain is treated via organcrosstalk or via corresponding dermatomes.
 18. The method of claim 1,wherein said pain is non-infectious pain.
 19. The method of claim 18,wherein said non-infectious pain is acute non-infectious pain or chronicnon-infectious pain.
 20. The method of claim 3, wherein said probioticcomposition treats symptoms of a urinary tract infection.
 21. Acomposition comprising a probiotic composition formulated for treatingpelvic pain.